Antipsychotics possess anti-glioblastoma activity by disrupting lysosomal function and inhibiting oncogenic signaling by stabilizing PTEN

The repurposing of medications developed for central nervous system (CNS) disorders, possessing favorable safety profiles and blood-brain barrier permeability, represents a promising strategy for identifying new therapies to combat glioblastoma (GBM). In this study, we investigated the anti-GBM activity of specific antipsychotics and antidepressants in vitro and in vivo. Our results demonstrate that these compounds share a common mechanism of action in GBM, disrupting lysosomal function and subsequently inducing lysosomal membrane rupture and cell death. Notably, PTEN intact GBMs possess an increased sensitivity to these compounds. The inhibition of lysosomal function synergized with inhibitors targeting the EGFR-PI3K-Akt pathway, leading to an energetic and antioxidant collapse. These findings provide a foundation for the potential clinical application of CNS drugs in GBM treatment. Additionally, this work offers critical insights into the mechanisms and determinants of cytotoxicity for drugs currently undergoing clinical trials as repurposing agents for various cancers, including Fluoxetine, Sertraline, Thioridazine, Chlorpromazine, and Fluphenazine.

The oligos used to generate each guideRNA are listed in the supplemental material.Cells were transfected at a MOI of approximately 10. Stable cells were established using puromycin selection for 7 days.The following guideRNAs were deigned to target luciferase (control) and PP2A subunits.

Intracranial Xenografts
Approximately 1x10 5 cells (3 µL) were intracranially injected into the brain of athymic nu/nu female mice (6-8 weeks old).Mice implanted with U87-MG cells were treated with 10 mg/kg perphenazine for 5 days followed by 2 days off, starting eight days post implantation.Mice implanted with U87-MG-PTEN cells received ten total treatments of 10 mg/kg perphenazine for 5 days followed by 2 days off, starting eight days post implantation.Mice were monitored for weight loss or severe neuro-symptoms until moribund, at which point they were sacrificed.

Bioluminescence Imaging
Mice implanted with U87-MG-Luc2 cells were intraperitoneally injected with a luciferin (PerkinElmer: 122796) solution (15 mg/ml in PBS, dose of 150 mg/kg).Bioluminescence images were acquired using an IVIS Lumina system and analyzed by Living Image software.

MTS Viability Assays
Cells were seeded in a 96-well plate at a concentration of 1x10 4 cells per well.
Drugs/controls were added 24 hours after plating.48 hours later, 20 µL/well MTS reagent (abcam: ab197010) was added to each well, incubate for 1 hour at 37 °C in standard culture conditions and measured.

Immunofluorescence
About 1.5x10 4 cells were plated per well in an 8-well chamber slide and incubated overnight.Cells were washed 2x with ice-cold PBS and fixed with 4% formaldehyde for 10 minutes.Cells were washed 2x with ice-cold PBS and permeabilized using 0.5% triton X-100 in PBS.Cells were incubated in blocking buffer (5% horse serum, 2% fish gelatin) for 60 minutes at 37 °C.Primary antibody was added in blocking buffer and incubated overnight at 4 °C on a rocker.Slides were washed 3x with PBST (0.1% Tween 20) and Invitrogen IgG (H+L) Highly Cross-Adsorbed Secondary Antibody was added at 1:1000 in blocking buffer.Cells were incubated for 1 hour at 37 °C and washed 1x with PBST.
Slides were mounted with ProLong Diamond Antifade Mountant with DAPI (Invitrogen: P36971) and sealed.Imaging was conducted on Cytation 5 imager.

NAD + /NADH Quantification
NAD + /NADH quantification was performed using the NAD + /NADH Quantification Kit (Sigma: MAK037).Approximately 2x10 5 cells were seeded per well in a 6-well plate.Cells were incubated with drugs for 24 hours prior to cell lysis.Cells were pelleted, washed with PBS, and lysed in 500 µL extraction buffer.We homogenized the lysates using ten rounds of sonication (1s, 10% amplitude, with a 30s interval, Branson 450 Digital Sonifier).250 µL of each sample was aliquoted and incubated in a heating block at 60 °C for 30 minutes to decompose NAD + .For each sample, 50 µL of total (NAD + & NADH) and NADH alone (decomposed NAD + ) were plated in triplicate along with the provided NADH standard.100 µL reaction mix containing the NAD + cycling enzyme was added to all samples for 5 minutes to convert NAD + to NADH.Subsequently, we added 10 µL of the NADH developing reagent to each sample and incubated at room temperature for 90 minutes.
Absorbance was read using a Cytation 5 imager (Biotek).NAD + levels were determined by subtracting NADH from decomposed samples from total NADH.

Seahorse XF Real-Time ATP Rate Assay Kit
We assessed glycolytic and mitochondrial ATP production using the Seahorse XF Real-Time ATP Rate Assay Kit (Agilent: 103592-100).10,000 cells were plated per well in a 24-well plate, 24 hours prior to starting the assay. 1 hour before starting the experiment, the cell culture media was changed out with Seahorse XF DMEM medium (Agilent: 103680-100) containing 10 mM glucose, 1 mM pyruvate, 2 mM glutamine, and desired vehicle or drugs.The assay was run on an XFe24 analyzer using the standard Seahorse XF Real-Time ATP Rate Assay protocol (20 min: 1.5 µM oligomycin; 40 min: 0.5µM Rotenone/antimycin A).

Triple Quad LC-MS/MS
Metabolites were quantified using the multiple reactions monitoring method (MRM) by selecting the appropriate qualifier and quantifier using a Triple Quad LC-MS/MS (Agilent).
The source parameters were as follows: drying gas temperature 325 °C, drying gas flow 11 L/min, nebulizer gas pressure 45 psi, and capillary voltage 4000 V.The MS scan was fixed at 100-1000 mass-to-charge ratio (m/z), and the data scan speed was fixed at 1spectra per second for data acquisition.Samples was injected on a reverse-phase C18 stable bond column (2.1 x 50 mm x 1.8 µm) (Agilent: 827700-902).Mobile phase consisted of water containing 0.1% acetic acid (A) and ACN containing 0.1% acetic acid (B).The samples were resolved for 12.5 min at a flow rate of 0.4 mL/min and the column